.
Impact of Duffy Antigen Receptor for Chemokine (DARC)-null
polymorphism on T cell function and pro-inflammatory cytokine responses during HIV-1 infection
Submitted by: Zesuliwe Barbrah Shangase (209503819) Supervisor: Dr. Christina Thobakgale-Tshabalala
Submitted in partial fulfilment of the requirements for the degree of Master of Medical Sciences
In the School of Laboratory Medicine and Medical Sciences at the Discipline of HIV Pathogenesis Programme,
Nelson R. Mandela School of Medicine, College of Health Sciences, University of KwaZulu-Natal, Durban
2018
i PREFACE
The experimental work described in this thesis was conducted at the HIV Pathogenesis Programme Immunology laboratory, Doris Duke Medical Research Institute, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, from February 2017 to November 2018, under the supervision of Dr Christina Thobakgale- Tshabalala.
This work has not been submitted in any form for any degree or diploma to any tertiary institution.
Z. Shangase Date 04 December 2018
C.F. Thobakgale-Tshabalala Date 04 December 2018
ii
PLAGIARISM: DECLARATION
I, Zesuliwe Shangase, declare that
(i) The research reported in this dissertation, except otherwise indicated, is my original work.
(ii) This dissertation has not been submitted for any degree or examination at any other university.
(iii) This dissertation does not contain other person’s data pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons.
(iv) This dissertation does not contain other person’s writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then:
(a) Their words have been re-written but the general information attributed to them has been referenced.
(b) Where their exact words have been used, their writing has been placed inside quotation mark and referenced.
(v) Where I have reproduced a publication of which I am not the author, co-author or editor, I have fully referenced such publications.
(vi) This dissertation does not contain text, graphics or table copied and pasted from internet unless specifically acknowledged, and the source being detailed in
dissertation and in the references section.
Signed Date: 04 December 2018
iv
RESEARCH OUTPUT
Awarded a full scholarship based on abstract merit to present poster at the 8th Infectious Diseases in Africa (IDA) Symposium, 12-17th November 2018. Cape Town, South Africa.
Oral and Poster Presentations
Zesuliwe Shangase, Kewreshini Naidoo, Thumbi Ndungu’ and Christina Thobakgale- Tshabalala. Impact of Duffy antigen receptor for chemokine (DARC)-null polymorphism on T cell function and pro-inflammatory cytokine activity during HIV-1 infection. Poster presentation, November 2018. IDA Symposium, Cape Town, South Africa.
Shangase ZB, Naidoo K, Ndung’u T, Thobakgale-Tshabalala CF. Impact of Duffy antigen receptor for chemokine (DARC)-null polymorphism on T cell function and pro-inflammatory cytokine activity during HIV-1 infection. School of laboratory Medicine and Medical Sciences, Oral presentation, 29 August 2018. Durban, South Africa.
v
DEDICATION
This thesis is dedicated to my loving husband Ndumiso Jule for his unconditional love, support, and constant encouragement and our son Avuyile Jule for his patience.
vi
ACKNOWLEDGEMENTS
I would like to express my gratitude to all those who made it possible for me to complete this study.
Firstly, I would like to thank God for his immense blessings and for providing me the strength and courage to complete this study;
Dr. Christina Thobakgale-Tshabalala, my supervisor and mentor, for her time, patience, guidance and support throughout the course of the research.
Prof Thumbi Ndung’u, my co-supervisor for his academic guidance and support.
Kewreshini Naidoo for assistance with laboratory training and results analysis.
Akeem Ngomu and Nontlantla Mdletshe for their kind assistance with laboratory work.
Staff and students from HPP for their patience and support.
I am grateful to National Research Foundation (NRF) and South African Medical Research Council (SAMRC) for providing me with funding during the course of my studies.
vii
TABLE OF CONTENT
Contents
PREFACE ... i
PLAGIARISM: DECLARATION ... ii
RESEARCH OUTPUT ... iv
DEDICATION... v
ACKNOWLEDGEMENTS ... vi
TABLE OF CONTENT ... vii
ABBREVIATIONS ... x
LIST TABLES ... xiii
LIST OF FIGURES ... xiv
ABSTRACT ... xvi
CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ... 1
1.1 HIV/AIDS epidemic ... 1
1.1.1 HIV/AIDS global epidemic ... 1
1.1.2 HIV/AIDS in Sub-Saharan Africa ... 2
1.2 HIV Virology ... 3
1.2.1 The Classification and structure ... 3
1.2.2 HIV-1 Life cycle ... 5
1.2.3 Assembly and budding ... 7
1.3 The natural response to HIV-1 infection... 8
1.4 Brief overview of the host immune responses to HIV infection ... 10
1.4.1 Innate responses ... 10
1.4.2 Humoral immune response ... 11
1.5 CD4+ T cell responses to HIV-1 infection ... 12
viii
1.6 CD8+ T cell responses to HIV-1 infection and their role in Acute and Chronic
HIV-1 infection ... 13
1.7 Role of neutrophils in immune response ... 15
1.8 Duffy-null genotype and neutropenia ... 16
1.10 Aims and Objectives of the study... 23
CHAPTER 2: MATERIALS AND METHODS ... 24
2.1 Study Participants ... 25
2.2 CD4+ T cell count, viral load and polymorphonuclear cells (PMNs) measurements ... 25
2.3 DARC -46T→C SNP genotyping ... 26
2.4 Isolation of peripheral blood mononuclear cells (PBMC) and counting ... 26
2.4.1 Cryopreservation of PBMCs ... 28
2.4.2 Thawing of cryopreserved PBMCs ... 28
2.5 Flow cytometry assay ... 29
2.5.1 Phenotypic characterization of CD8+ T cells ... 30
2.5.2 Assessment of T cell function using Intracellular Cytokine Staining (ICS) ... 31
2.5.3 Assessment of T cell proliferation using carboxyfluorescein diacetate, succinimidyl ester (CFSE) ... 34
2.6 Luminex Assays Principle ... 35
2.6.1 Luminex for detection of neutrophil and T cell specific cytokines ... 37
2.7 Statistical analysis ... 39
CHAPTER 3: RESULTS ... 40
3.1 Clinical characteristics of study participants ... 40
3.2 Levels of CD8+ T cell activation and exhaustion differ by HIV status and not by DARC status ... 42
3.3 Gag and Envelope specific CD8+ T cell activity in HIV infected and HIV uninfected individuals with or without DARC polymophism ... 48
3.4 Increased T cells proliferation with HIV-1 infection and not by DARC status ... 52
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3.5 Decreased IL-8 and MIP1-β and increased G-CSF plasma levels in HIV infection.
... 53
CHAPTER 4: DISCUSSION ... 56
REFERENCES ... 64
APPENDICES ... 77
Appendix A ... 77
Appendix B ... 81
Appendix C ... 84
Appendix D ... 87
x
ABBREVIATIONS
AIDS Acquired immune deficiency syndrome APC Antigen Presenting Cell
ARG Arginase
ART Antiretroviral therapy
CCL5 Chemokine (C-C motif) ligand 5 CCR5 Chemokine Receptor 5
CD107a Cluster of Differentiation 107a
CD4+ T cells Human T cells expressing CD4+ antigen CD8+ T cells Human T cells expressing CD8+ antigen CD38 Cluster of Differentiation 38
CD57 Cluster of Differentiation 57 CDK6 Cyclin-dependent Kinase 6 CFR Circulating Recombinant Forms CSF3 Colony Stimulating Factor 3
CFSE Carboxyfluorescein succnimidyl ester CTL Cytotoxic T lymphocyte
CXCL2 Chemokine (C-X-C motif) ligand 2 DARC Duffy Antigen Receptor for Chemokine
DC Dendritic Cell
DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid
ELISA Enzyme-linked Immunosorbent Assay Env Envelope
ER Endoplasmic Reticulum FBS Fetal Bovine Serum FCS Fetal Calf Serum
FMO Fluorescence Minus One Gag Group-specific antigen
G-CSF Granulocyte Colony Stimulating Factor GM-CSF Granulocyte-Macrophage Colony Stimulating H2O2 Hydrogen Peroxide
HAART Highly active antiretroviral treatment
xi HIV Human immunodeficiency virus HLA Human leukocyte antigen
HLA-DR Human leukocyte antigen DR isotype ICS Intracellular cytokine staining
IFN-γ Interferon gamma IL-1β Interleukin 1 beta IL-2 Interleukin 2 IL-4 Interleukin 4 IL-5 Interleukin 5 IL-6 Interleukin 6 IL-7 Interleukin 7 IL-10 Interleukin 10 IL-12 Interleukin 12 IL-13 Interleukin 13 IL-17 Interleukin 17
MCP-1 Monocyte Chemoattractant Protein 1 MHC Major histocompatibility complex
MIP-1β Macrophage Inflammatory Protein 1 beta mRNA Messenger ribonucleic acid
Nef Negative regulatory factor NETs Neutrophil Extracellular Traps NC Nucleocapsid (p7)
NK Natural killer P24 Capsid protein
PBS Phosphate buffered saline
PBMC Peripheral blood mononuclear cells PD-1 Programmed Death 1
PD-L1 Programmed Death Ligand 1 PHA Polyhydroxyalkanoate PMN Polymorphonuclear Pol Polymerase
RBC Red Blood Cell
Rev Regulator of virion expression protein RNA Ribonucleic acid
xii ROS Reactive Oxygen Species RT Reverse Transcriptase SA-PE Streptavidin-phycoerythrin SEB Staphylococal enterotoxin B SIV Simian immunodeficiency virus Tat Transactivator of transcription
TB Tuberculosis
TCR T cell receptor
TNF-a Tumor necrosis factor alpha UNAIDS United Nation on AIDS Vif Viral infectivity factor Vpr Viral protein R
Vpu Viral protein U
WHO World health organization
xiii LIST TABLES
Table 1.1 Global and regional reports of HIV-1 epidemic (UNAIDS, 2018) ... 1 Table 1.2: Prevalence of benign neutropenia by ethnic populations (Thobakgale and Ndung’u, 2014).
... 17 Table 3.1 Clinical characteristics of study participants ... 40 Table 2.1: Table showing flourochrome conjugated antibodies, supplier and volume of antibodies used per well. ... 33 Table 3.1: Clinical characteristics of study participants ... 40 Table 2.1 Table showing flourochrome conjugated antibodies, supplier and volume of antibodies used per well. ... 33
Table 1.1 Global and regional reports of HIV-1 epidemic (UNAIDS, 2018) ... 1 Table 1.2: Prevalence of benign neutropenia by ethnic populations (Thobakgale and Ndung’u, 2014).
... 17 Table 3.1 Clinical characteristics of study participants ... 40
xiv
LIST OF FIGURES
Figure 1.1: The Global number of people living with HIV on Antiretroviral Therapy for 2010 to 2015
(UNAIDS, 2018). ... 2
Figure 1.2: The map showing HIV-1 prevalence by the end of 2017 (UNAIDS, 2018). ... 3
Figure 1.3: The structure of HIV. The viral capsid is enclosed by lipid membrane with the integration of viral glycoprotein and surface proteins. Within virion is the viral capsid made up of a pair of single stranded RNA molecules. The capsids contain viral genome and enzymes (Rockstroh, 2011). ... 4
Figure 1.4: Genomic organization of HIV-1. Gag, pol and envelope are shown in rectangles (black, orange and green respectively). Tat, rev, nef, vif, vpr, vpu are represented in squares (red, yellow, blue, light green, and white respectively) LTR (long terminal region) is shown in grey (Rockstroh, 2011). ... 5
Figure 1.5: Replicative cycle of HIV-1. The HIV virus attaches and enters the cell. 2) Uncoating takes place. 3) Reverse transcription takes place where viral RNA is converted into viral DNA. 4) The integration of Viral DNA into the host genome. 5) The infected cell from the host produces copies of the viral RNA. 6) The host cell assembles new viruses and the newly assembled viruses leave the cell to infect other cells (iBase, 2017). ... 7
Figure 1.6 HIV clinical course. Diagram of typical course of HIV-1 infection showing changes in CD4+ and CD8+ T-cell counts in peripheral blood and plasma viral load, and three phases of HIV infection (Munier and Kelleher, 2007). ... 9
Figure 1.7: Schematic representation of CD8+ T cell recognition. Processed viral peptides from HIV- 1 infected cells are presented by MHC class I proteins to CD8+ T cell for recognition and killing (wikiwand). ... 13
Figure 1.8: Global prevalence of Duffy-null genotype (Howes et al., 2011). ... 18
Figure 1.9: Mechanisms involved in T cell inhibition (left panel) and activation (right panel) by neutrophils (Leliefeld et al., 2015). ... 21
Figure 1.10: A simplified model of how neutrophil counts in people of African descent may affect susceptibility or ability to control diseases. (a) In the context of neutropenia, caused by genetic or environmental factors, neutrophils fail to clear infection and there may be inability to prime T cells, which might increase risk of infection and pathogen spread. (b) When neutrophils count and functions are optimal as dictated by genetic and other undetermined factors, there is increased pathogen clearance, and neutrophil priming and cross-talk with T cells result in HIV-1 infection (Thobakgale and Ndung’u, 2014). ... 22
Figure 2.1: Image showing PBMC Ficoll density gradient separation technique. ... 27
Figure 2.2: Image showing LSRFortessa flow cytometer (BD Bioscience, USA) (A) and (B) flow diagram showing the work flow from each run to data that can be used for statistical analysis. ... 29
Figure 2.3: Illustration of cell division and CFSE intensity as each cell divides ... 34
Figure 2.4: Diagram of Bio-plex sandwich immunoassay ... 36
Figure 2.5: Diagram showing steps in Luminex assay. ... 37
Figure 3.1: CD4 and polymorphonuclear cells (PMN) counts in HIV uninfected individuals and HIV-1 infected participants and stratified by DARC status within the groups. ... 42
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Figure 3.2: Gating strategy for the identification of CD8+ T cells and measurement of activation and exhaustion markers from bulk PBMCs by multicolour flow cytometry from an HIV infected participant.. ... 44 Figure 3.3: Frequencies of CD38+ HLA-DR+ CD8+ T cells in HIV-1 negative individuals and HIV-1 positive individuals. ... 45 Figure 3.4: : Frequencies of PD-1+ CD8+ T cells in HIV-1 negative and HIV-1 positive individuals. ... 46 Figure 3.5: Frequencies of CD57+ CD8+ T cells in HIV-1 negative individuals and HIV-1 positive individuals. ... 46 Figure 3.6: The correlations of PMNs and frequencies of CD38+, HLA-DR+, PD-1+ and CD57+ on CD8+ T cells in HIV negative and HIV positive individuals. ... 47 Figure 3. 7: Representative gating strategy for the identification of CD8+ T cells from cells from PBMCs by multicolour flow cytometry.. ... 48 Figure 3.8: Detection of intracellular cytokines following PBMC stimulation. ... 50 Figure 3.9: Detection of intracellular cytokines following PBMC stimulation.. ... 51 Figure 3.10: Gating strategy for proliferative capacity of CD8+ T cells upon stimulation with Gag peptide pools using multicolour flow cytometry. ... 52 Figure 3.11: Measurement of CD8+ T cell proliferation using CFSE. ... 53 Figure 3.12: Summary of measured cytokine and chemokine concentration levels. ... 54 Figure 2.1: Image showing PBMC Ficoll density gradient separation technique after whole blood was centrifuged for 30 minutes (Higdon et al., 2016). ... 27 Figure 2.2: Image showing LSRFortessa flow cytometer (BD Bioscience, USA) (A) and (B) flow diagram showing the work flow from each run to data that can be used for statistical analysis. ... 29 Figure 2.3: Illustration of cell division and CFSE intensity as each cell divides (Luzyanina et al., 2007) ... 34 Figure 2.4: Diagram of Bio-plex sandwich immunoassay (Bio-Rad, USA) showing how magnetic beads, capture antibody, detection antibody, and streptavidin combine to make sandwich of the biomarker of interest (Bio-Rad, USA). ... 36 Figure 2.5: Diagram showing steps in Luminex assay. Step 1, magnetic beads with biomarker of interest are added, step 2, detection antibody is added and step 3 is the addition of streptavidin (Bio- Rad. USA). ... 37
xvi ABSTRACT
The Duffy-null trait presents as non-expression of the Duffy antigen receptor for chemokines (DARC) on red blood cells and is highly prevalent in African populations. The Duffy-null genotype is the most significant genetic determinant of ethnic neutropenia and has been previously associated with HIV-1 transmission and disease progression. A recent role of the involvement of neutrophils in cross-talk with other immune cells such as natural killer (NK) and cytotoxic T cells has been reported, however these cells are impaired following HIV-1 infection. Some studies have suggested that neutrophils are important in priming of T cells and in mediating pro-inflammatory responses, however, little is known about the impact of DARC- null trait-associated neutropenia on T cell function. We here investigated the association of Duffy-null trait with T cell phenotype and function and proinflammatory cytokine activity in HIV-1 infection.
Antiretroviral therapy (ART) naïve chronically HIV-1 infected individuals (n=20) were recruited from Prince Mshiyeni hospital and HIV uninfected donors (n=20) were from a cohort of young women in Umlazi township in KwaZulu Natal, Durban. Multicolor flow cytometry was used to measure T cell activation (CD38 and HLA-DR expression) and exhaustion (PD-1 expression) in HIV-1 positive and HIV uninfected individuals. T cell function (IFN-γ, TNF-α and CD107a expression) following stimulation with HIV peptides (gag and envelope) was measured by intracellular cytokine staining (ICS) and T cell proliferative capacity by carboxyfluorescein succinimidyl ester (CFSE) was measured following stimulation with gag peptide pool. Lastly, we measured pro-inflammatory cytokine and chemokine plasma levels (G-CSF, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, MCP-1, MIP-1β, and TNF-α) using Luminex.
Our results showed highly activated CD8+ T cells as measured by HLA-DR and CD38 expression in HIV-1 positive individuals compared to HIV uninfected donors (p<0.0001). Our findings also showed high CD8+ T cell responses in HIV-1 infected patients compared to HIV uninfected donors (p=0.02 for CD107a and p=0.03 for IFN-γ) as well as high proliferative capacity of CD8+ T cells in HIV-1 positive patients compared to uninfected donors (p=0.05).
Our data showed no significant differences between CD4+ T cell activation and exhaustion in HIV-1 positive individuals and HIV uninfected donors. No differences were also observed in
xvii
in cytokine production and replicative capacity in CD4+ T cells between HIV-1 positive individuals and HIV uninfected donors.
Multiplex data showed higher G-CSF levels in HIV-1 infected patients compared to HIV uninfected individuals (p=0.04). Unexpectedly, the levels of proinflammatory cytokine IL-8 and chemokine MIP-1β were higher in the HIV-1 negative group compared to HIV-1 positive individuals (p<0.0001 and p=0.0008 respectively). All these findings in T cells and proinflammatory cytokine expression were not significantly different upon stratification by DARC status.
Collectively, our data suggest that HIV-1 infection results in CD8+ T cell activation and exhaustion with increased cytolytic activity. These data also suggest that despite neutropenia, Duffy-null individuals may have developed evolutionary mechanisms to compensate for possession of this genetic trait with regard to how the immune system responds to infection.
1
CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW
1.1 HIV/AIDS epidemic
1.1.1 HIV/AIDS global epidemic
Human immunodeficiency virus 1 (HIV-1) was discovered three and half decades ago yet still remains a life-threatening disease globally. HIV-1 infections were estimated to be 36.9 million throughout the world with 1.8 million new HIV-1 infections, including 940 000 thousands AIDS related deaths by the end of 2017 (UNAIDS, 2018). According to the 2018 global report, at least 2.2 million new infections were observed globally in 2010, however this number has now reduced to 1.8 million by the end of 2017 (Table 1.1). As seen in Figure 1.1, the number of people living with HIV-1 with access to antiretroviral therapy (ART) continues to rise; 7.5 million HIV-1 infected people were on ART in 2010 compared to 19.5 million by the end of 2016 (Avert, 2018, WHO, 2018).
Even though the number of people having access to ARVs has increased, the goal of completely eradicating HIV-1 has not been reached.
Table 1.1 Global and regional reports of HIV-1 epidemic (UNAIDS, 2018)
2
Figure 1.1: The Global number of people living with HIV on Antiretroviral Therapy for 2010 to 2015 (UNAIDS, 2018).
1.1.2 HIV/AIDS in Sub-Saharan Africa
Developing countries are heavily affected by the HIV-1 epidemic, accounting for 66% of HIV-1 infections. A heavier burden of these infections is in Africa, with sub-Saharan Africa (SSA) being the most heavily impacted region with 19.6 million in 2017 (Figure 1.2). In SSA, there were 800 000 reported new HIV-1 infections with 380 000 deaths due to AIDS related causes by the end of 2017, with only 12.9 million of the population on antiretroviral therapy (UNAIDS, 2018). South Africa remains the epicentre of the HIV-1 epidemic, about 7.1 million people were HIV-1 infected by 2016; 270 000 people newly infected and 110 000 people died due to AIDS. At least 56% of the HIV-1-infected people in South Africa were on ART by the end of 2016 (Cornell et al., 2017). South Africa, KwaZulu-Natal (KZN) is the most affected province with a prevalence of 12.2%
(Council, 2017a) compared to Northern (Council, 2017b) and Western Cape (Council, 2017c) with prevalence of 6.8 and 5.6% respectively. UMgungundlovu District had a prevalence of 40.7% compared to seven out of nine Districts in KZN with a prevalence of 38.0% including Durban in 2012 (Kharsany et al., 2015).
3
Figure 1.2: The map showing HIV-1 prevalence by the end of 2017 (UNAIDS, 2018).
1.2 HIV Virology
1.2.1 The Classification and structure
HIV, the virus that causes AIDS, was first noticed in 1981 in homosexual men who died from unusual opportunistic infections and uncommon malignancies. HIV is a Lentivirus from a Lentiviridae family and is divided into HIV-1 and HIV-2 (Barré-Sinoussi et al., 1983). HIV-1 and HIV-2 were transmitted from primates to humans as simian immunodeficiency virus (SIV) (Sharp and Hahn, 2011). HIV-1 is most common in sub- Saharan Africa and worldwide, while HIV-2 is known to be common in Western Africa.
HIV-1 is divided into groups M, N, O and P. Group M is the HIV-1 strain that is further classified into nine clades (A, B, C, D, F, G, H, J, and K) and is the cause of global HIV- 1 outbreak.
In addition, when distinct subtypes combine their deoxyribonucleic acid (DNA) they form multipartite virus called circulating recombinant form (CRF) (Hemelaar, 2012). High HIV-1 replication rates, viral reverse transcriptase mutations and high recombination rates lead to genetic variability of HIV-1.
4
The diversity of the HIV-1 sequence makes it a challenge for the immune system to control HIV-1 replication as well as develop an effective HIV vaccine.
Structurally, HIV-1 has a diameter of 119 to 207 nanometre (nm) surrounded by lipoprotein membrane and it has enveloped ribonucleic acid (RNA) virus (Goodsell, 2015, Briggs and Kräusslich, 2011). HIV-1 consist of two strands of RNA packaged within a core of viral proteins for integrity and it is also surrounded by a lipid bilayer (Figure 1.3), in addition to RNA strands, the capsid itself is also composed of viral proteins p24, p6 and p7 (Levy, 1993). The structure also contains enzymes (reverse transcriptase (RT), protease and integrase) to develop new virions. Surrounding the capsid is the matrix, which is composed of viral protein p17. Matrix is surrounded by phospholipid bilayer derived from the host cell. It is also composed of two viral glycoproteins: gp41, the fusogenic protein that pierces the envelope and combines the inside of the virus to the outside, and gp120, the protein on the outside that binds CD4+
T cells (Fanales-Belasio et al., 2010, Gelderblom et al., 1987, Montagnier, 1985).
Figure 1.3: The structure of HIV. The viral capsid is enclosed by lipid membrane with the integration of viral glycoprotein and surface proteins. Within virion is the viral capsid made up of a pair of single stranded RNA molecules. The capsids contain viral genome and enzymes (Rockstroh, 2011).
5
There are nine genes of the HIV-1 virus (Figure 1.4), these can be divided into different types. The structural proteins include Gag, Env and Pol; the regulatory proteins are Tat and Rev, and Vpu, Vif, Vpr and Nef that are the accessory genes (Levy, 1993). Tat and Rev are expressed early in the virus cycle and are responsible for enhancement of viral replication. Vpu, Vif, Vpr and Nef differentiate HIV-1 from other retroviruses, regulate antiretroviral responses, and increase viral replication (Sivro, 2014).
Figure 1.4: Genomic organization of HIV-1. Gag, pol and envelope are shown in rectangles (black, orange and green respectively). Tat, rev, nef, vif, vpr, vpu are represented in squares (red, yellow, blue, light green, and white respectively) LTR (long terminal region) is shown in grey (Rockstroh, 2011).
1.2.2 HIV-1 Life cycle
HIV-1 is a retrovirus, with an outer envelope and two copies of RNA and reverse transcriptase that will convert RNA to DNA. There are 6 steps of HIV-1 replication;
binding and entry, HIV replication and integration, assembly, budding and release of new mature virions.
6 1.2.2.1 Virus attachment and entry
During this process, the virus places itself on the cell surface and uses CD4 T cell receptors to prepare for fusion. Viral fusion is caused by binding of the virus to target cell membrane and this is mediated by gp41 (Weiss, 1993). This is followed by binding of gp120 and CD4 receptors located on the surfaces of T helper cells, macrophages, monocytes and dendritic cells (Fauci, 2007b). This is then followed by a conformational change of the envelope exposing specific domain of gp120 which results in the binding of the C-C chemokine co-receptor 5 (CCR5) (Clapham and McKnight, 2002).
The function of these chemokine co-receptors is to facilitate homing of different types of immune cells to the site of infection (Alkhatib and Berger, 2007). The stock of the gp120 and 41 pieces through into the host cell undergoes conformational change unfolding itself, drawing two membranes together and the binding of the co-receptors triggers pore formation allowing the formation of a helical bundle structure which completes the fusion of the virus and cellular membranes, (Figure 1.5) (Klasse, 2012, Gomez and Hope, 2005).
1.2.2.2 Reverse transcription and Uncoating
Fusion is followed by uncoating where the viral genetic material (two viral RNA strands and three essential replication enzymes) is injected essentially into the target cell and the envelope protein is left at the cell surface. The virus matrix and capsid proteins are digested when the virus enters the cell (Bukrinsky et al., 1993). This then releases viral enzymes and viral RNA into the cell cytoplasm. Reverse transcription takes place when the reverse transcriptase (RT) takes the viral RNA and by using host nucleotide, converts that viral RNA into a single strand of DNA. This single stranded DNA is again transcribed into double stranded DNA (Figure 1.5).
1.2.2.3 Integration
Following reverse transcription, integrase enzyme transports the double stranded DNA and carries it through a nuclear pore into the nucleus of the cell. Integrase then mediates integration of viral DNA to host genome. At this point, the viral DNA is referred to as
‘provirus’. The provirus may stay inactive for longer periods or undergo transcription and translation for new viral proteins (Bukrinsky et al., 1993).
7
Figure 1.5: Replicative cycle of HIV-1. The HIV virus attaches and enters the cell. 2) Uncoating takes place. 3) Reverse transcription takes place where viral RNA is converted into viral DNA. 4) The integration of Viral DNA into the host genome. 5) The infected cell from the host produces copies of the viral RNA. 6) The host cell assembles new viruses and the newly assembled viruses leave the cell to infect other cells (iBase, 2017).
1.2.3 Assembly and budding
Host genome at this stage contain HIV-1 genetic material. RNA polymerase then comes along and make messenger RNA (mRNA). These mRNAs encode for different viral proteins. They associate with ribosomes at the surface of rough endoplasmic reticulum (ER). Messenger RNA then makes envelope protein directly produced into the ER, shuttled through ER, and taken to the cell surface where it becomes embedded to cell membrane and colonise with other envelope proteins produced (Freed, 2001).
8
At the same time, there are other mRNAs being produced that allow translation of other viral proteins. Tat and Rev proteins are also synthesized. Tat binds to the TAR site at the beginning of the HIV-1 RNA within the nucleus and stimulates the transcription and the production of longer RNA transcripts. The function of Rev is to facilitate the transcription of longer RNA transcripts and the expression of structural and enzymatic genes. In addition, Rev also inhibits the synthesis of regulatory proteins, promoting the formation of mature viral particles (Fanales-Belasio et al., 2010). These mature viral particles are then released by budding from the host cell surface to continue the cycle of HIV-1 replication.
1.3 The natural response to HIV-1 infection
Immediately after HIV-1 infection, the virus replicates in the mucosal cells, submucosal cells as well as in draining lymphoreticular tissues (Emau et al., 2006, Weiss et al., 2010), and at this time, the virus is unable to be detected in plasma (Keele et al., 2008).
Despite of the route of viral transmission and the first cells infected, within a few days, viral replication occurs on the gastrointestinal tract in gut-associated lymphoid tissue (Brenchley et al., 2004, Mehandru et al., 2004, Veazey et al., 1998). In this tissue, the majority of infected cells are resting CD4+ T cell which lack activation markers and express low levels of CCR5, a chemokine receptor found on T cell surfaces (Li et al., 2005) and other cells including macrophages and Langerhans cells (Derdeyn and Silvestri, 2005). During initial infection, epithelial cells of the mucosa recruit dendritic cells and dendritic cells secrete cytokines to attract activated CD4+ T cells to site of infection (Haase, 2010).
After infection, early immune responses rapidly drives viral replication since the presence of the virus recruits other T cells to the site of infection which in turn lead to more T cells being infected (Li et al., 2009).
During the acute phase of infection, the HIV-1 plasma viral load goes up exponentially and CD4+ T cells are depleted in the first weeks of infection, this phase is also associated with a responsive immune system trying to fight the infection (Figure 1.6) (Fanales- Belasio et al., 2010).
9
Figure 1.6 HIV clinical course. Diagram of typical course of HIV-1 infection showing changes in CD4+ and CD8+ T-cell counts in peripheral blood and plasma viral load, and three phases of HIV infection (Munier and Kelleher, 2007).
Few weeks after infection, the virus replicates at low levels, developing latent infection (Figure 1.6) (Fauci, 2007a). Latent stage lasts for years, however, some people may progress faster because of the failure of immune response to fight the infection due to persistent viral replication. The virus remains untraceable for years in other patients which indicates that they are able to control the infection (Fanales-Belasio et al., 2010).
Following this, HIV-1 spreads to lymphoid tissues leading to further immune destruction and development of opportunistic infections like tuberculosis (TB) and pneumonia (Ford et al., 2009, McMichael et al., 2010b) (Figure 1.6).
The last stage of HIV-1 infection is AIDS where the immune system is totally damaged.
At this stage the numbers of CD4+ T cells are very low, below 200 cells per/mm3, also, during this stage there is increased chances of opportunistic infections (Akram and Inman, 2012, Brooks et al., 2009) and eventually leads to death (Figure 1.6). HIV-1 related mortality and morbidity have decreased since the initiation of ART. After ART initiation, the viral load decreases, however, CD4 T cell counts are not consistent.
10
Suppression of viremia decreases immune activation and that lowers HIV-1 replication preventing risk of transmission (Yamashita et al., 2001). Despite the effectiveness of ART and improved life expectancy in HIV infected individuals, there are still challenges faced in the new ART era; for example, the occurrence of non-AIDS co-morbidities (UNAIDS, 2018).
1.4 Brief overview of the host immune responses to HIV-1 infection 1.4.1 Innate responses
Skin and the mucosal membrane are physical barriers that act as the first line of defence against invading microbes. Mucosal membrane has mucous that traps invading pathogens. There are also epithelial cells that provide the first line of defence by blocking pathogens from entering the host. However, some pathogens such as HIV-1 can cross the physical barriers and upon doing so they encounter host immune responses where it can be eliminated by natural killer cells and CD8+ T cells (Medzhitov and Janeway, 2000).
The innate immune response is very rapid after pathogen invasion. It also includes cells like neutrophils, macrophages, monocytes, dendritic cells (DCs), natural killer cells (NK cells) and other cell types. Neutrophils, monocytes, and macrophages have phagocytic effects. They engulf infected cells, invading pathogens or dying cells. On the other side, DCs are antigen-presenting cells (APC) that take up the antigen and present it to specific cells depending on the captured antigen. Dendritic cells regulate the adaptive immune response and they bring both arms of the immune system together.
1.4.1.1 Natural killer cells
Natural killer (NK) cells are the main effector cells of the innate immune response. They synthesise and release perforin which is a protein that kills targeted cells by opening pores on their surfaces (Chang and Altfeld, 2010). Natural killer cells bridge the innate and adaptive immune systems in that they directly respond to viruses, develop memory-like responses after initial pathogen encounter or vaccination, and they shape the adaptive immune response of the infection which helps them to clear the infection faster when it comes back (O’sullivan et al., 2015).
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The increased sub-population of NK cells in HIV -1 infection has been shown, however these cells are not functional enough to clear the virus (Alter et al., 2005). This impaired functionality is thought to be caused by viral products, suggesting that HIV-1 might have evolved to escape mechanisms against NK cell mediated control (Jost and Altfeld, 2012).
1.4.1.2 Dendritic cells (DCs)
Dendritic cells are in the skin, mucosa as well as in lymphoid tissues. These cells are known to be professionals in terms of presenting antigens to cells such as T cells and NK cells capable of killing pathogens (Vivier et al., 2008, Dustin and Long, 2010). Their important function is to take up the antigen, process it and present it to T or NK cells to create memory as well as prevent these T or NK cells from destroying self-antigens. They do this by expressing antigenic peptides that associates with major histocompatibility complex class II in T cells which recognize these antigens and build immune synapses.
Immune synapses are stable intercellular junctions between the cells. To add, DCs also secrete cytokines for regulation of the immune responses (Monks et al., 1998). In HIV-1 infection, the major targeted cells are CD4+ T cells, however DCs as antigen presenting cells, represent a crucial subset in HIV-1 infection by presenting HIV-1 to target cells (Miller, 1998).
1.4.2 Humoral immune response
Humoral immunity is an antibody-mediated immune response. Antibodies are proteins produced by plasma cells and protect the host from infection firstly by neutralization, opsonization and lastly to kill by phagocytosis and activating the complement cascade (Casadevall, 2018). In HIV-1 infection, the humoral immune response antibodies block the virus from entering and infecting target cells (McMichael et al., 2010a). Antibody responses develop four to eight weeks after infection and are principally targeted against virions that are free-floating, whilst some antibodies may eliminate HIV-1 infected cells.
Most antibodies cannot prevent the transmission of HIV-1 from one cell to another. Most people infected with HIV-1 develop antibodies to the virus however, a very small number do not, possibly because of individual immune dysfunctions (Alter and Moody, 2010, Ellenberger et al., 1999).
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The antibody response is often directed towards the Env variable region of the infecting viral strain. Mutation of the envelope proteins (gp120 and gp41) make it possible for the virus to escape from antibodies (Aasa-Chapman et al., 2004). The rate at which HIV-1 mutate is so high that B cells fail to keep up with mutations and fail to produce enough antibodies and eventually get exhausted. HIV-1 can evade humoral response by selecting escape mutants which are not easily neutralized (Wei et al., 2003). Neutralizing antibodies only arise several months following infection (Aasa-Chapman et al., 2004, Burton, 1997).
Even though memory B cells can produce wide range of antibodies to previously encountered virus, there is an impaired response to new antigens due to mutations of the virus.
1.5 CD4+ T cell responses to HIV-1 infection
CD4+ T cells bring together different processes of the adaptive immunity and provide help to B cells and CD8+ T cells (Porichis and Kaufmann, 2011b), however CD4+ T cells are highly targeted by HIV. The HIV-1 specific CD4+ T cell responses are high during acute phase of infection, but these responses decrease after few months of HIV-1 infection (Streeck and Nixon, 2010) and the numbers of CD4+ T cells go down because of increased viral replication within these cells exposes them to death.
As much as the responses of CD4+ T cells are high in many people infected with HIV-1, their magnitude is lower than CD8+ T cell responses (Betts et al., 2001). Stimulation of CD4+ T cells starts when APCs present the processed antigen to CD4+ T cells through HLA class II molecules. Naïve CD4+ T cell requires a co-stimulatory signal to be activated such as the binding of CD28 protein to CD4+ T cells. Activated CD4+ T cells differentiate into T helper 1 (Th1) cells, T helper 2 (Th2), Th17, T follicular helper cells (TFH) and regulatory T cells (Tregs).
T helper 1 cells produce IL-2 for CD8+ T cell proliferation and differentiation (Oxenius et al., 2000), Th2 produce IL-4 and IL-5 to further stimulate B cell proliferation (Zhu et al., 2010) and Th17 cells have been shown to be reduced in the gut-associated lymphoid tissue (GALT) and blood during HIV-1 infection and can predict disease progression (Brenchley et al., 2008, Falivene et al., 2015).
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Reduction of CD4+ T helper cells during HIV-1 infection has been reported to affect all immune responses to HIV-1 including CD8+ T cell responses (Porichis and Kaufmann, 2011a).
1.6 CD8+ T cell responses to HIV-1 infection and their role in Acute and Chronic HIV-1 infection
For CD8+ T cells to respond to virus, they recognize HLA class I molecule encoded by the MHC locus on the surface of the target cell. HLA molecule and CD8 molecule interaction is caused by the binding of the epitope-HLA complex to the T cell receptor (TCR). This interaction activates the killing of the infected cells either by lytic or non- lytic mechanisms (Figure 1.7).
Figure 1.7: Schematic representation of CD8+ T cell recognition. Processed viral peptides from HIV-1 infected cells are presented by MHC class I proteins to CD8+ T cell for recognition and killing (wikiwand).
The principal mechanism in which the CTL kill virus infected cell is through the secretion of granzymes and perforin (Shankar et al., 1999). Perforin open pores on the surface of the target cell allowing granzyme to enter the cell and destroy viral proteins, and the target cell eventually dies through apoptosis (Figure 1.7) (Hudig et al., 1993).
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The second mechanism in which CTLs kill virus infected cells is calcium independent Fas mediated pathway. Here the TCR interacts with target cell Fas ligand (FasL) on the surfaces of target cells (Wong et al., 1997) which then triggers the apoptosis cascade then death of the target cell (Broere et al., 2011).
The non-lytic pathway involves soluble molecules secreted by CD8+ T cells to neutralize infected cells. This pathway does not directly kill HIV-1 infected cell but plays a huge role in controlling viral replication (Chang et al., 2003). The CD8+ T cells secrete suppressive factors like β-chemokines which inhibits viral replication by binding to the cognate receptors which blocks viral access to co-receptors important for viral binding and entry into target cells (Copeland, 2002).
Other molecules produced by CD8+ T lymphocytes such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) play a huge role in regulating the host immune responses against HIV-1 infection (Cao et al., 2003, Roff et al., 2013).
Immune responses by CD8+ T cells have been detected in early stages of HIV-1 infection (Fiebig et al., 2003). During acute infection, HIV-1 replication increases exponentially in the peripheral blood then moves to lymphoid tissues such as lymph nodes (Kahn and Walker, 1998). As described in the above sections, this persistent replication of the virus activates HIV-1 virus-specific CTL responses. Several studies have shown the effectiveness of CTLs in HIV-1 infection (Koup et al., 1994a, Borrow et al., 1994, Deng et al., 2015, Lv et al., 2014). In addition, in vivo studies done on non-human primates have shown that removal of CD8+ T cells after they were infected with SIV led to increased viral load (Schmitz et al., 2005). This suggest that HIV-1 specific CD8+ T cells play an important role in controlling HIV-1. Other studies had shown that, the appearance of CTLs correlated with low plasma viral load (Koup et al., 1994b). Previous studies also reported that the increased numbers of CTLs specific for HIV-1 in acute infection had an effect in controlling viral load (Ndhlovu et al., 2015b). Even though these studies suggest that CTLs can control HIV-1 virus, these responses are narrowly directed towards certain epitopes and have low magnitude (Altfeld et al., 2001, Dalod et al., 1999, Radebe et al., 2011).
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In some cases, the HIV-1 viral activity is strong that they escape CTL responses (Borrow et al., 1997), leading to loss of CTL function and disease progression (Lichterfeld et al., 2004b) despite an increase in the breadth of these responses (Alter et al., 2002, Cao et al., 2003).
In chronic HIV-1 infection, HIV-1 specific CD8+ T cells do not proliferate because of the continuous exposure to the virus that lead to their exhaustion. This is further supported by a study done by Betts and Harari 2008, where they looked at the CD8+ T cell proliferation aspect in HIV-1 viremic controllers with protective HLA class I alleles and found that CD8+ T cells were able to proliferate compared to chronic HIV-1 infected individuals (Betts and Harari, 2008, Koofhethile et al., 2016)
In addition, because of ongoing HIV-1 replication, CD8+ T cells become exhausted and their function is lost. Studies have reported that programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway is responsible for the exhaustion of anti-viral CD8+ T cells during chronic infections. CD8+ T cell exhaustion in HIV-1 infection is associated with increased expression of CD57 and PD-1 on the surfaces of T cells (Day et al., 2006).
Binding of PD-1 to its ligand PD-L1 expressed on myeloid cells including neutrophils, downregulate proliferation and production of effective cytokines such as IFN-γ, TNF-α and IL-2 by T cells (Keir et al., 2008, Bowers et al., 2014). Impaired functions of virus- specific T cells and B cells leads to immune dysfunction (Palmer et al., 2014).
1.7 Role of neutrophils in immune response
Neutrophils also known as polymorphonuclear leukocytes (PMNs), are a group of leukocytes that control infection and inflammation. They are the first cells to be recruited to the site of infection by inflammatory factors released by macrophages and mast cells, in response to pathogen invasion or danger from infected and necrotic cells. Neutrophils act as the first line of defence against pathogens and as mediators of other immune cells.
These cells are short-lived (8–12 hours in the circulation and 1–2 days in tissues) and important cells for both innate and adaptive immune systems (Borregaard, 2010).
Neutrophils originate from precursor myeloid cells in the bone marrow, here they become myeloblasts before maturing into neutrophils.
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During infection, which may include HIV-1 infection, neutrophils are activated and release neutrophil extracellular traps (NETs), undergo degranulation, and secrete reactive oxygen species (ROS) such as H2O2 to fulfil their functions (Amulic et al., 2012, Mócsai, 2013). Although evidence suggests that neutrophils induce human T cell responses through activation of dendritic cells (Kalyan and Kabelitz, 2014), other studies suggest that neutrophils also supress T cell activation by releasing arginase-1, which impairs the T cell responses by blocking the availability of L-arginine. Arginine is involved in wound healing, proliferation of T cells and production of TCR ζ chain that is important in the activation of T cells (Pillay et al., 2013, Rodriguez et al., 2007, Rodriguez et al., 2002).
ROS production by neutrophils modifies host molecules, resulting in a weak response to stimuli and eventually apoptosis or phagocytosis by macrophages (Perskvist et al., 2002, Amulic et al., 2012). During HIV-1 infection, chemo attractants released by neutrophils are lost and are then fully restored during HAART (Younas et al., 2016).
1.8 Duffy-null genotype and neutropenia
Given the importance of neutrophils as the first line of defence against pathogens and mediators of other immune cells, when the number of these cells in the blood circulation is reduced, it can lead to severe immunodeficiency. Neutropenia is a condition that results in a decrease in circulating absolute neutrophils counts in the blood to below 1500 cells/µl in children older than one year and adults of any ethnic group (Haddy et al., 1999).
It is characterized clinically as mild (neutrophil counts between 1000 cells/µl and 1500 cells/µl), moderate (500–1000 cells/ml) or severe (below 500 cells/µl) (Newburger, 2016, Thobakgale and Ndung’u, 2014, Haddy et al., 1999, Paz et al., 2011). Individuals with absolute neutrophil count less than 200 cells/µl undergo severe or even fatal infections (Newburger, 2016). Neutropenia can either be congenital or benign. Congenital neutropenia is normally found in Caucasians and these people often experience bacterial infections due to lack of adequate number of neutrophils (Hsieh et al., 2010).
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Benign neutropenia is the type of neutropenia where serious infections do not occur even with neutrophil counts below 200 cells/µl (Klein, 2009, Hsieh et al., 2010). Benign ethnic neutropenia has been found to be common in 25-50% of persons of African descent, and is not associated with clinical outcome compared to other ethnic groups (Haddy et al., 1999, Grann et al., 2008, Shoenfeld et al., 1985). The prevalence of benign ethnic neutropenia reported in different ethnic groups is summarized in Table 1.2.
Table 1.2: Prevalence of benign neutropenia by ethnic populations (Thobakgale and Ndung’u, 2014).
The causes of benign ethnic neutropenia are poorly understood because there is no deficiency in granulocyte forming cells; there is normal myeloid maturation however there is reduced release of mature neutrophils from the vascular endothelium and bone marrow to the periphery (Paz et al., 2011, Reiner et al., 2011).
Even though causes of benign neutropenia are not known, an identified genetic determinant for benign neutropenia has been implicated. Studies have linked benign ethnic neutropenia to Duffy-null genotype (Reich et al., 2009). Using admixture mapping, a region in chromosome 1 was found to be the one that account for differences in white blood cell counts between African Americans and European Americans (Nalls et al., 2008).
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It was subsequently confirmed that single nucleotide polymorphism, (rs2814778, - 46T→C) of Duffy antigen receptor for chemokines (DARC), that is identified on chromosome Iq22 of the receptor in red blood cells (RBCs) is predictive of neutrophil count in African Americans (Reich et al., 2009). Also there are reports that DARC null trait is highly prevalent in Africa (Figure 1.8) (Howes et al., 2011).
Figure 1.8: Global prevalence of Duffy-null genotype (Howes et al., 2011).
DARC encodes for Duffy antigen on red blood cells, vascular endothelial and neuronal cells. It acts as a sink for pro-inflammatory chemokines, regulating their levels in circulation, affecting neutrophil localization, chemotaxis and migration (Lee et al., 2003, Paz et al., 2011). The DARC null polymorphism selects for DARC null phenotype in which DARC is not expressed on red blood cells. This trait is associated with neutropenia and explains the variations of neutrophil counts between African-Americans and European-Americans (Reich et al., 2009). This is supported by a previous study that confirmed that approximately 59% of African-American males had DARC-null genotype compared to 65% of men and women from South Africa (Julg et al., 2009b). Apart from DARC gene polymorphisms in African Americans, other genetic variants associated with low white blood cells have been discovered. These include genetic variants in CXCL2 on chromosome 4, near CDK6 on chromosome7, and CSF3 on chromosome 17 (Reiner et al., 2011). People with HIV-1 infection suffer from neutropenia (Levine et al., 2006).
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Before the association between DARC-null trait and neutropenia was shown, a study by He and colleagues reported that DARC-null might play a role in HIV acquisition and disease progression. It was suggested that DARC-null phenotype was associated with a 40% increased chance of HIV-1 acquisition and may account for up to 11% of HIV-1 burden in Africa. These authors also showed that following HIV-1 infection, DARC-null trait was associated with slower disease progression (He et al., 2008).
The findings by He et al were contradicted by another study done by Walley and others (2009) who demonstrated that, the DARC-null trait had no effect on HIV-1 acquisition or disease progression (Walley et al., 2009). The following year Julg et al, did a study where the effect of the DARC-null genotype on the outcomes of disease progression were examined. The study assessed DARC null, heterozygous and wild-type genotypes and found that these had no effect on CD4 decline or viral load among DARC genotypes, suggesting that if DARC-null trait is associated with disease progression, it was independent of these factors (Julg et al., 2009a). In addition, DARC null has evolved on the African continent as a resistant factor to malaria by preventing invasion on red blood cells by Plasmodium vivax (Horuk et al., 1993). However, the impact of this polymorphism on infectious diseases like HIV-1 remains largely unknown.
There are mechanisms proposed to explain how DARC presence influences acquisition and disease progression. Before onset of infection, HIV-suppressive chemokines (CCL5, a chemotactic cytokine for T cells that suppresses HIV-1 replication) associated with DARC-expressing erythrocytes may act as a protective shield on the DARC receptor, preventing HIV-1 to attach to the red blood cell and be transferred to HIV-1 target cells.
In this case DARC-null trait increases HIV-1 acquisition risk. However, following HIV- 1 acquisition, HIV-1 can bind to DARC receptor on the surface of RBCs thereby allowing transfer of the virus to target cells. This indicates that DARC-expressing RBCs might act as carriers of infectious HIV-1 particles to susceptible cells such as CD4+ T lymphocyte leading to increased disease progression (He et al., 2008).
Furthermore, DARC-positive individuals may be predisposed to a more pro- inflammatory state that promotes HIV-1 replication and spread (Kulkarni et al., 2009).
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It is hypothesized that people with neutropenia may experience slow disease progression because of reduced inflammation due to less HIV-1 particles that can bind to DARC-null in red blood cells (Thobakgale and Ndung’u, 2014).
1.9 Interaction of T cells and neutrophils and role in HIV-1 infection
Neutrophils have emerged as important regulators and have been suggested to play a role in cross talk with immune cells such as natural killer cells, B cells, T cells, macrophages, dendritic cells and platelets (Boudaly, 2009, Costantini and Cassatella, 2011, Scapini et al., 2008, Silva, 2010). Neutrophils have been reported to prime CD8+ T cells in the bone marrow and lymphoid organs such as lymph nodes through antigen presenting cells (Maletto et al., 2006). Neutrophils recruit local macrophages to the site of infection and these neutrophils then release IL-1β and TNF-α to recruit more T cells into the infected areas (Hampton and Chtanova, 2016).
Duffy and others (2012) reported that chemokines released by neutrophils also attract T cells to the site of infection. Upon T cells arrival, neutrophils release IFN-γ, TNF-α, cathepsin G, and neutrophil elastase to differentiate T cells. Neutrophils also secrete IL- 12 to activate T cells (Duffy et al., 2012b).
Previous studies speculated that neutrophils might inhibit T cells responses in viral infections. The mechanisms of this suppression involve reactive oxygen species (ROS), arginase-I (ARG), and PD-L1 (de Kleijn et al., 2013, Highfill et al., 2010, Schmielau and Finn, 2001).
The first mechanisms in which neutrophils affect survival of T cells is the inhibition mechanism (Figure 1.9). Here, neutrophils undergo degranulation and release serine proteases to inhibit T cell proliferation by stimulating IL-2 and IL-6 and promoting the shedding of IL-2 and IL-6 receptors on the surface of T cells. Neutrophils also inhibit T cell proliferation by the release of reactive oxygen species (ROS) and Arginase-1 as described in the above sections that arginase-1 and ROS downregulate T cell receptor chain (TCRζ) on T cells, which in turn lead to T cell arrest at G0-G1 phase (Figure 1.9).
Neutrophils also express PD-L1 to induce T cell apoptosis by binding to interferon- dependent PD-1 that is expressed on T cells (Leliefeld et al., 2015).
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The second mechanism involves the activation of T cells by neutrophils. Here, neutrophils activate T cells by presenting antigen to them directly in that, when neutrophils undergo apoptosis after ingesting pathogen, dendritic cells take up antigen from neutrophils and present it to T cells.
Indirect presentation involves presentation of antigen to T cells using MHC class I. An alternate pathway involves the ingestion of antigen and release of bacterial products by neutrophils to activate γδ-T cells (Leliefeld et al., 2015).
Figure 1.9: Mechanisms involved in T cell inhibition (left panel) and activation (right panel) by neutrophils (Leliefeld et al., 2015).
In HIV-1 infection, the priming of HIV-1 virus-specific T-cells by neutrophils has been reported (Duffy et al., 2012b). A previous study has shown that neutrophils in the blood of HIV-1-infected individuals express high levels of PD-L1 induced by HIV-1 virions.
This study also showed that neutrophil PD-L1 levels correlated with the expression of PD-1 and CD57 on CD4+ and CD8+ T cells (Bowers et al., 2014).
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This previous study also highlighted that neutrophils purified from the blood of HIV-1- infected patients suppress T cell function via several mechanisms including PD-L1/PD-1 interaction and production of reactive oxygen species (ROS) (Bowers et al., 2014).
The gathered data suggest that chronic HIV-1 infection results in an induction of immunosuppressive activity of neutrophils characterized by high expression of PD-L1 and an inhibitory effect on T cell function. However, the impact of DARC-null on T cell function in Africans with HIV-1 infection is unknown. Studies are needed to understand the role of the DARC-null genotype and low neutrophil counts on various diseases to identify possible biomarkers of disease protection or severity. These could also motivate identification of race-specific reference ranges and therapies for people of African descent to prevent poor outcomes of different illnesses.
We here expanded on the proposed model by Thobakgale and Ndung’u, in 2014 (Figure 1.10), on how neutrophil counts in Africans may affect susceptibility or ability to control diseases (Thobakgale and Ndung’u, 2014) by investigating the role of DARC null on T cell function in Africans from Durban, KwaZulu-Natal.
Figure 1.10: A simplified model of how neutrophil counts in people of African descent may affect susceptibility or ability to control diseases. (a) In the context of neutropenia, caused by genetic or environmental factors, neutrophils fail to clear infection and there may be inability to prime T cells, which might increase risk of infection and pathogen spread. (b) When neutrophils count and functions are optimal as dictated by genetic and other undetermined factors, there is increased pathogen clearance, and neutrophil priming and cross-talk with T cells result in HIV-1 infection (Thobakgale and Ndung’u, 2014).
23 1.10 Aims and Objectives of the study
Here we characterized the mechanisms and the impact that DARC null trait might have on patients’ CD8+ T cell profiles and proinflammatory cytokine and chemokine responses. The aims and objectives of the study were as follows:
1. Aim 1: To investigate the role of DARC-null linked neutropenia on T cell phenotype during HIV-1 infection.
Objective 1: To assess the T cell activation, senescence and exhaustion (using HLA-DR and CD38, CD57 and PD-1 respectively) using multi-parameter flow cytometry in HIV infected individuals and HIV uninfected donors with and without the DARC-null polymorphism using flow cytometry.
2. Aim 2: To investigate the role of DARC-null linked neutropenia on T cell proliferation and function during HIV-1 infection.
Objective 2: To assess the T cell proliferation and cytokine (CD107a, IFN-γ and TNF- α) production following antigen stimulation (gag and envelope) in HIV-1 infected individuals and HIV uninfected donors with and without the DARC-null polymorphism using ICS.
3. Aim 3: To investigate the role of DARC-null linked neutropenia on systemic inflammation during HIV-1 infection
Objective 3: To assess whether differences in the pro-inflammatory cytokines (G-CSF, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL- 17, MCP-1, MIP-1β, and TNF-α ) levels exist in DARC-positive and DARC-negative HIV uninfected and infected individuals using Luminex assay.
Hypothesis
We hypothesised that the Duffy-null genotype will negatively affect T cell function, phenotype and proinflammatory cytokine levels in people of African descent with HIV- 1 infection.
24 1.11 Candidate’s contribution
The candidate did the experiments, analysis and interpretation of results and write-up.
Blood collection was done at clinic sites and sample processing was done at the HPP laboratories.
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CHAPTER 2: MATERIALS AND METHODS
2.1 Study Participants
In this study we recruited young HIV-1 positive females (n=19) and males (n=1) with median age of 23 years (range 20-24) from the HIV Pathogenesis Programme (HPP) Acute study cohort at Prince Mshiyeni Memorial Hospital, located at Umlazi township, Durban, KwaZulu-Natal, South Africa. The precise time of infection for these participants was unknown but were in the chronic stage of infection for this cross sectional analysis and were antiretroviral therapy (ART) naïve.
In addition, young HIV uninfected individuals (n=20), all women with a median age group of 21 (range 19-22) years were recruited from the Females Rising through Education, Support, and Health (FRESH) cohort located in Umlazi township, Durban, KwaZulu-Natal, South Africa. These women otherwise healthy, however at very high risk of HIV-1 infection, were recruited and closely monitored for HIV infection of as part of HIV prevention curriculum, empowerment, life skills offered through the FRESH study.
This pilot study was exploratory and a sub-study within the HPP studies that received ethical clearance; reference number BF131/11 for the FRESH cohort (for HIV uninfected individuals) and E036/06 for the HPP acute cohort that recruited HIV-1 infected participants. Sample calculation for this exploratory pilot sub-study was not needed.
Written informed consent was obtained from each individual for participation in the study. This sub-study was approved by Biomedical Research Ethics Committee (BREC) of the University of KwaZulu –Natal under ethics approval number BE346/17.
2.2 CD4+ T cell count, viral load and polymorphonuclear cells (PMNs) measurements
Absolute CD4 counts were measured using the Tru-Count technology using flow cytometry as previously described (Zulu et al., 2017). Plasma viral loads for the HPP acute cohort were measured using Nuclisens HIV-1 QT assays, the Biomerieux Easy- MAG and Biomerieux Easy-Q platform for nucleic acid extraction with a detection limit of 400 HIV-1 RNA copies/ml plasma according to manufacturer’s instructions (Dong et
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al., 2018). Sysmex XN system (Sysmex, Canada) was used to measure neutrophil count using full blood count test.
2.3 DARC -46T→C SNP genotyping
A single nucleotide polymorphism DARC T-46C (rs2814778) was genotyped using TaqMan allelic discrimination assays in all subjects as previously described (Julg et al., 2009a, Malkki and Petersdorf, 2012) and the genotypes were verified by direct sequence analysis of samples. Briefly, DNA was isolated from buffy coats (EDTA tubes) using QIAamp DNA Blood Midi Kit from Qiagen. The DNA was then Nano-dropped and standardised at concentration of 50ng/μl with nuclease free water (Ambion). This was then followed by preparation of master mix (Taqman universal PCR master mix, SPN assay mix and nuclease free water). Target sequences were then amplified, and analysis was done on Light Cycler 480 II (Roche). Participants with two copies of C allele (CC) were labelled Duffy null, and those with one allele or none (TC/TT) were labelled Duffy positive.
2.4 Isolation of peripheral blood mononuclear cells (PBMC) and counting
Peripheral blood mononuclear cells (PBMCs) are populations of immune cells such as lymphocytes (T cells, B cells and natural killer cells), monocytes and dendritic cells and were isolated from whole blood by Ficoll density gradient centrifugation. In brief, PBMCs were isolated, frozen and stored as follows:
Whole blood samples were collected from donors using sodium heparin (BD Vacutainer, USA) and were processed within 4 hours of collection. Before processing, reagents were put at room temperature and were wiped with 70% ethanol before being placed inside the class II Biological Safety Cabinet (BSC). Blood samples were poured into 50ml conical tubes for PBMC processing. A 50ml tube of whole blood was then centrifuged at 500xg for 10 minutes at room temperature. After the 10 minutes spin, the supernatant which was plasma, was carefully removed and stored at -80 ºC ultra-freezers. The remaining whole blood was diluted with equal amount of Dulbeco’s Phosphate Buffered Saline (DPBS) (Life Technologies, UK) that contained 1% of penicillin-streptomycin solution and was gently mixed by inverting. Diluted blood was overlaid into a 50ml conical tube separation medium (15ml of Histopaque-1077, Sigma Aldrich) at room temperature.
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The tube was centrifuged for 30 minutes at 500xg at room temperature. After this, whole blood was divided into different cell layers as seen in Figure 2.1.
Figure 2.1: Image showing PBMC Ficoll density gradient separation technique after whole blood was centrifuged for 30 minutes (Higdon et al., 2016).
A sterile pipette was used to remove the lymphocytes (buffy coat layer) from the interface and transferred to another sterile 50ml conical tube, followed by addition of DPBS to wash the lymphocytes (cells were centrifuged at 500xg for 10 minutes at room temperature and this step was done twice). After the second wash was complete, the supernatant was decanted, and the lymphocyte pellet was suspended in 20 mL of R10 (RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, 100 U/ml penicillin, 1.7Mm sodium glutamate and 1% of Hepes buffer).
